1. Field of the Invention
The present invention pertains to the field of biomass processing to produce fuels, chemicals and other useful products and, more specifically, to saccharifying lignocellulosic biomass materials to produce sugars for conversion to ethanol and other products with enhanced Glycosidases, such as cellulase and xylanase efficacy through selective binding or blocking of the lignin component. Use of a protein wash enhances bioconversion efficiency by increasing the availability of cellulase and other enzymes to cellulose.
2. Description of the Related Art
Cellulosic biomass is useful for generating ethanol. Such materials specifically known as lignocellulosic materials, or biomass, (e.g. wood and solid wastes), have been used as source material to generate carbohydrates, which in turn may be used to produce ethanol, as well as other products.
Lignocellulosic biomass is a complex structure of cellulose fibers wrapped in a lignin and hemicellulose sheath. The ratio of the three components varies depending on the type of biomass. Typical ratios are as follows:
TABLE 1SOFT-CORNCORNWOODSCOBSRDF*STOVERCELLULOSE42%40%52%37%HEMICELLULOSE25%36%26%22%LIGNIN28%13%20%17%OTHER 5%11% 2%24%*RDF—REFUSE DERIVED FUEL FROM MUNICIPAL WASTE SYSTEMS
Table 1 is only an approximation. For example, wood differs in composition, depending on the particular type of wood, where softwoods (gymnosperms) generally have more glucomannans and less glucuronoxylans than do hardwoods.
Cellulose is a polymer of D-glucose monomer with β-1-4-linkages between each monomer forming chains of about 500 to 10,000 D-glucose units. Hemicellulose is a polymer of sugars, primarily D-xylose with other pentoses and some hexoses, also with β-1-4-linkages. Lignin is a complex random polyphenolic polymer. Lignocellulose biomass represents an inexpensive and readily available substrate for the preparation of sugars. These sugars may be used alone, fermented to produce alcohols and industrial chemicals, or chemically converted to other compounds.
Ethanol is one of the alcohols that may be produced using carbohydrate derived from a lignocellulosic biomass, and has a number of industrial and fuel uses. Of particular interest is the use of ethanol as a gasoline additive that boosts octane, reduces pollution, and partially replaces gasoline in fuel mixtures. Ethanol-blended gasoline formulations are well-known commercial products commonly called “gasohol”. It has been proposed to eliminate gasoline almost completely from the fuel and to burn ethanol in high concentrations.
Conversion of cellulose biomass into renewable fuels and chemicals often involves chemical and/or enzymatic treatment of the biomass with cellulase or other enzymes. In particular, cellulase enzymes hydrolyze cellulose to D-glucose, which is a simple sugar. In high lignin content lignocellulosic biomass, high doses of cellulase are needed to degrade the cellulose with high yields because the lignin binds preferentially with the cellulase, thereby reducing access of cellulase to cellulose. Consequently, when processing high lignin content biomass materials, less cellulase is available to degrade cellulose because the lignin coating of the cellulose fibers scavenges cellulase. Thus, the effectiveness of the process for digesting cellulose is reduced.
Bioconversion of cellulose biomass to ethanol has been studied since the 1940's. However, the cellulose-to-ethanol process is not yet economical compared to producing petroleum products by existing technology. Enzymatic hydrolysis is a fairly slow process. The costs of cellulases are high, and the required amount of cellulases is also high, which increases processing costs. Reduction in the amount of cellulase needed to obtain a satisfactory sugar yield can have a significant impact on process economics. Therefore, improving the efficiency of enzyme use is a major need in the bioconversion process.
The mechanism of hydrolysis and the relationship between the structure and function of various cellulases have been extensively studied. Several factors are thought to influence enzymatic hydrolysis of cellulose. These factors include lignin content, hemicellulose content, acetyl content, surface area of cellulose and cellulose crystallinity. It is generally understood that the lignin present in complex substrates, such as steam-exploded wood, especially softwoods, has a negative effect on cellulase activity. The exact reasons are poorly understood because the complexity of biomass is such that reducing one barrier to digestion can enhance or disguise the importance of others. For example, cellulose hydrolysis has been shown to improve with increasing lignin removal, although differences are reported in the degree of lignin removal that is needed, as well as the physical form of the lignin.
A variety of factors may be associated with the deleterious effects of lignin upon saccharification. The ratio of syringyl moiety to guaiacyl moiety in the lignin may affect saccharification. Although the exact role of lignin in limiting hydrolysis has been difficult to define, one probable significant limitation is the effect of lignin on fiber swelling and the resulting influence on cellulose accessibility. The removal of lignin increases accessibility of cellulose and allows more cellulase activity. This is problematic in that some lignin complexes are physically and chemically resistant to enzymatic attack. While some lignin components are water soluble, others are insoluble and may precipitate from solution. Condensed lignin has the ability to adsorb protein from aqueous solutions. Lignin removal may open more surface area for enzymatic attack and reduce the amount of cellulase that is non-specifically adsorbed on the lignocellulosic substrate. Studies involving acid pretreated softwood report a positive correlation between digestibility and the extent of delignification, but the results are complicated by the presence of hemicellulose. Some substrates require higher temperatures for hemicellulose removal to be effective; suggesting that hemicellulose is not the only additional factor impacting digestibility and other evidence does not support a role for hemicellulose in changing cellulose digestibility.
Although cellulose crystallinity is generally reasoned to impede enzymes, rates slow with increasing crystallinity in some studies, but increase in other studies. The degree of crystallinity may not significantly change over an extended hydrolysis time. Crystallinity seems less important than lignin removal and impacts saccharification rates more than yields. Several studies have focused on explaining cellulose digestibility by the accessibility of cellulose to enzymes. Correlations have been developed to relate rates to pore volume and accessible surface area. However, the complex shape of cellulases may create difficulty in penetrating such pores, and concerns have been raised about substrate changes during these measurements. Additionally, most measurement techniques measure gross surface area and may include non-specific adsorption, e.g., onto lignin.
Cellulases are often utilized as a mixture of enzymes having different activities, and the enzyme structures differ between microorganisms that express enzymes of a given family. While the mechanisms of hydrolysis and the relationship between the structure and function of various cellulases have been extensively studied, many details of enzymatic activity are still poorly understood. The enzymatic hydrolysis of cellulose substrates is strongly affected by end-product inhibition and enzyme features. Low specific cellulase activity on cellulose is an important factor that limits the effectiveness of hydrolysis. One way to circumvent this low specific activity is to recycle and reuse the enzyme. However, non-productive cellulase adsorption plays an important role in the development of ways to reuse enzymes and affects recycle efficiency.
Besides the complexity of the different types of cellulases, activity on the substrate is also complicated by substrate characteristics. Due to resistance from the complex structure and composition of natural cellulosic biomass, the lignocellulose substrate should be pretreated to make it as susceptible as possible to the action of the enzymes. Many pretreatment methods have been developed. For example, increased accessibility of lignocellulose substrate can be achieved by solubilizing hemicellulose in harsh acidic conditions.
Cellulase adsorption on lignocellulosic substrates containing high content of natural materials has not been extensively studied. Typically, lignocellulosic substrates contain a much higher content of lignin compared to “model” cellulose substrates. Lignin may inhibit enzymatic hydrolysis of lignocellulosic material. Cellulases are not only adsorbed to the cellulosic part of the substrate, but are also adsorbed to the lignin. Lignin not only shields the cellulose but also acts as a competitive adsorbent. However, lignin does not appear to restrict the extent of hydrolysis of the carbohydrate moiety if sufficient cellulase is present. Cellulolytic enzymes bind strongly to lignin. When adsorption profiles are compared, much more enzyme protein is associated with hydrolyzed residues of lignocellulosic materials than that of model cellulose. For example, β-glucosidase has a high affinity for various lignin fractions, while it does not bind to polysaccharides.
Generally, lignin may play an important role in enzymatic hydrolysis of lignocellulosic material (Sutcliffe & Saddler, Biotechnol. Bioeng. Symp. 8th, 17:749-62 (1986);). It has been shown that the enzymes are not only adsorbed to the cellulosic part of the substrate, but also bind strongly to lignin (Boussaid et al, Optimization of hemicellulose sugar recovery from a steam-exploded softwood, Proceedings of the Biomass Conference of the Americas, 3rd, Montreal, Aug. 24-29, 1997): Chemoglazov et. al., Enzyme Microb. Technol., 10:503-507 (1988); Deshpande, M. V. and K. -E. Eriksson, “Reutilization of enzymes for saccharification of lignocellulosic materials,” Enzyme and Microbiol. Technology, 6: 338-340, (1984); Sutcliffe & Saddler, Biotechnol. Bioeng. Symp. 8th, 17:749-62 (1986)). Specially, β-glucosidase appears to have a high affinity for various lignin fractions while it does not bind to polysaccharides (Sutcliffe & Saddler, Biotechnol. Bioeng. Symp. 8th, 17:749-62 (1986). The inactivation of cellulases by lignin has been reported (Avgerinos, G. C. and D. I. C. Wang, “Selective solvent delignification for fermentation enhancement,” Biotechnology and Bioengineering, 25(1): 67-83, (1983); Excoffier, G., B. Toussaint, et al. “Saccharification of Steam-Exploded Poplar Wood.” Biotechnology and Bioengineering, 38(11): 1308-1317, (1991); Sutcliffe & Saddler, Biotechnol. Bioeng. Symp. 8th, 17:749-62 (1986). It appears that different types of lignin and forms of lignin may have influenced adsorption of cellulase components (Chemoglazov et al., Enzyme Microb. Technol., 10:503-507 (1988); Sutcliffe & Saddler, Biotechnol. Bioeng Symp. 8th, 17: 749-62 (1986). Previous work on the hydrolysis of cellulose has shown that hydrolysis of pretreated substrates is improved when proteins are present. For example, it is reported that lignin peroxidase blocks lignin binding in biomass to enhance ethanol yield from SSF (WO 94/29474 ). That BSA addition results in the same level of hydrolysis yield as increasing surfactant addition is also indicated by Eriksson (Eriksson, T., J. Borjesson, et al. “Mechanism of surfactant effect in enzymatic hydrolysis of lignocellulose,” Enzyme and Microbiol. Technology, 31(3): 353-364, (2002)). It is most likely that the lignin blocking effect of protein in lignocellulose hydrolysis is explained by the protein's ability to block the non-specific adsorption sites of the non-cellulose fraction of the substrate and enhance the amount of cellulase available to absorb on the cellulose fraction (Eriksson et al. 2002; Kawamoto, H., F. Nakatsubo, et al. “Protein-adsorbing capacities of lignin samples,” Mokuzai Gakkaishi, 38(1): 81-4, (1992); Zahedifar, M., F. B. Castro, et al. “Effect of hydrolytic lignin on formation of protein-lignin complexes and protein degradation by rumen microbes.” Animal Feed Science and Technology, 95(1-2): 83-92, (2002)). Of course, the mechanism of protein interaction with lignin to enhance enzymatic digestibility is an object of intense research and speculation.
Lignin plays an important role in enzymatic hydrolysis of lignocellulosic material, as reported in Sutcliffe & Saddler, Biotechnol. Bioeng. Symp. 8th, 17:749-62 (1986). Comparative adsorption profiles demonstrated that much more enzyme was retained with hydrolyzed residues, compared to that of model pure cellulose, as reported in Abdel & Saddler, Int. Conf. Biotechnol. Pulp Pap. Ind., 7th, C239-C242 (1998). In a study by Chemoglazov et. al., Enzyme Microb. Technol., 10:503-507 (1988), endoglucanases that adsorbed on lignin lost activity. The inactivating effect of lignin was observed also with steam-exploded substrate, but not if the latter was acid-treated, nor with the lignocarbohydrate complex. Sutcliffe et al., Biotechnol. Bioeng. Symp., 17: 749-762 (1986), report that adsorption of cellulases on different lignin preparations from steam-treated hardwood is influenced by the nature of the lignin and β-glucosidase was most affected by lignin. Thus, different types of lignin and forms of lignin may influence cellulase adsorption. Also, the form of the lignin, which contains distinct lignin and lignocarbohydrate complexes, seems to influence cellulases differently. It is generally agreed that the form and positioning of most lignin changes after steam-explosion, such that the lignin separates from cellulose to form agglomerates.
Several proposals have been made for solving the problem of ineffective and/or inefficient enzyme degradation of high lignin containing biomass materials. One of these is a pretreatment step that degrades or removes at least a portion of the hemicellulose and/or lignin from the biomass. For example, a combination of heat and acid pre-treatment of the lignocellulosic mass for a period of time has been used to hydrolyze hemicellulose. However, this process provides for only very limited removal of lignin, as reported in Grohmann et. al . . . Biotechnol. Bioeng. Symp. 17, Symp. Biotechnol. Fuels Chem., 8th, 135-151 (1986) and Torget et al., Applied Biochemistry and Biotechnology, 34-35:115-123 (1992).
Lignin removal from cellulosic fibers has also been proposed though using a caustic alkali, such as in Kraft pulping and paper making. However, this process does not produce simple sugars and does not separate the hemicellulose from the cellulose.
U.S. Pat. No. 4,668,340 issued to Sherman relates to biomass hydrolysis processing that produces almost exclusively hemicellulose sugars. Acid is introduced to the biomass, and is removed from each stage to be fed to the next in its sequence. The hydrolysis of cellulose is minimized in the process, and results in a cellulosic pulp containing over 90% of the feed α-cellulose.
U.S. Pat. No. 4,708,746 issued to Hinger relates to the specific hydrolysis of cellulose followed by treatment with high-pressure steam. However, the use of high steam alone does not provide for the complete hydrolysis of the cellulose substrate.
U.S. Pat. No. 5,125,977 issued to Grohmann et al., and U.S. Pat. No. 5,424,417 issued to Torget et al., relate to the prehydrolysis of a lignocellulosic biomass to solubilize the hemicellulosic sugars with concomitant release of some soluble lignin. Prehydrolysis renders the remaining cellulose more readily digestible with enzymes or other chemical means. U.S. Pat. No. 5,424,417 describes a process wherein lignocellulose is subjected to a prehydrolysis step by passing an acidic or alkaline solution through solid or lignocellulosic particles, with the continuous removal of soluble reaction products. The technique permits a less severe combination of pH, temperature, and time than conventional prehydrolysis. Extraction of hemicellulose and lignin occurs simultaneously in the same reactor and under the same conditions.
U.S. Pat. No. 6,022,419 issued to Torget et al. relates to a process in which a lignocellulosic biomass is fractionated by using a dilute acid, e.g., dilute sulfuric acid at 0.07 wt %, to convert cellulose into monomeric sugars in relatively high yields. However, cellulose hydrolysis using an acid catalyst is costly and requires special equipment. In addition, the desired sugars are labile in the harsh conditions, and significant amounts of unwanted and toxic by products typically form. If exposed too long, the glucose derived from the cellulose degrades into hydroxymethylfurfarol, which further degrades into unwanted degradation products including levulinic acid and formic acid. The acidic conditions similarly degrade xylose, which is formed from hemicellulose.
WO 94/29474 to Hinman relates to a process in which a treatment of lignocellulose minimizes binding of cellulase. A substrate is formed of cellulose, hemicellulose, and starch. A hydrolytic acid pretreatment agent is added to the substrate, as is a lignin peroxidase to block lignin binding sites in the biomass. Cellulase is added to the substrate using Simultaneous Saccharification and Fermentation (SSF) process conditions favorable for cell viability and conversion of ethanol.
Kadal et al., 53: 277-284 (1999), relates to the use of peroxide treatments to remove lignin under alkaline conditions during pulp bleaching. Under alkaline conditions, hydrogen peroxide reacts with both aliphatic and aromatic structures of lignin, leading to depolymerization and subsequent removal with water washing. Gould, Biotechnol. Bioeng., 26:46-52 (1984), reports the use of alkaline peroxide to remove lignin and improve enzymatic hydrolyzability of herbaceous residues. Ramos et al., Holzforschung 46:149-154 (1992), report the use of alkaline peroxide to steam explode hardwood. Yang et al., Biotechnology and Bioengineering 77(6): 678-684(2002), report the use of alkaline peroxide treatment to enhance the enzymatic digestibility of steam-exploded softwood substrates.
Generally, softwoods have been considered the worst-case scenarios as a feedstock for the bioconversion processes because their highly recalcitrant lignin reduces the efficiency of enzymatic hydrolysis. Schwald et al., Enzyme Systems for Lignocelluosic Degradation, Goughlan, M. P., Elsivier, N.Y., pp. 231-242 (1989), and Wu et al., Appl. Biochem. Biotechnol., 77-79, 47-54 (1998), report that a compromise in the pre-treatment conditions will likely be required, if softwood residues are to be considered as a potential feedstock for biomass processing, i.e., a medium severity process is needed between those optimized for high hemicellulose recovery and efficient cellulose hydrolysis.
According to the aforementioned pretreatment processes, cellulose substrates produced by pretreatment at medium severity (about log R0=3.76) contain a high lignin content that limits cellulase accessibility to cellulose. The term “R0” is used in the industry as an indicator of the relative severity of a treatment method for the processing of a biomass. Specifically, in the field of lignocellulosics and fractionation of wood components, “R0” has been used to define a “severity parameter.” This equation is described in Overend, R. P. & Chornet, E. (1987 Fractionation of lignocellulosics by steam-aqueous pretreatments. Phil. Trans. R. Soc. Lond., 523-36.):R0=t·exp[(T−100)/14.75]  (1)where R0 is the severity factor and is optimized at 3.8 for the prehydrolysis of hemicellulose, t is time of exposure in minutes, and T is temperature in degrees Centigrade.